Journal
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
Volume 97, Issue 9, Pages 3991-4002Publisher
SPRINGER
DOI: 10.1007/s00253-012-4508-z
Keywords
Agplectasin; STAMP; Plectasin; MRSA; Pichia pastoris
Categories
Funding
- National Natural Science Foundation of China [30771574, 30810303084, 30972125, 31001026]
- Beijing Natural Science Foundation [5062031, 5093030]
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A novel specifically targeted antimicrobial peptide (STAMP) that was especially effective against methicillin-resistant Staphylococcus aureus (MRSA) was designed by fusing the AgrD1 pheromone to the N-terminal end of plectasin. This STAMP was named Agplectasin, and its gene was synthesized and expressed in Pichia pastoris X-33 via pPICZ alpha A. The highest amount of total secreted protein reached 1,285.5 mg/l at 108 h during the 120-h induction. The recombinant Agplectasin (rAgP) was purified by cation exchange chromatography and hydrophobic exchange chromatography; its yield reached 150 mg/l with 94 % purity. The rAgP exhibited strong bactericidal activity against S. aureus but not Staphylococcus epidermidis or other types of tested bacteria. A bactericidal kinetics assay showed that the rAgP killed over 99.9 % of tested S. aureus (ATCC 25923 and ATCC 43300) in both Mueller-Hinton medium and human blood within 10 h when treated with 4x minimal inhibitory concentration. The rAgP caused only approximately 1 % hemolysis of human blood cells, even when the concentration reached 512 mu g/ml, making it potentially feasible as a clinical injection agent. In addition, it maintained a high activity over a wide range of pH values (2.0-10.0) and demonstrated a high thermal stability at 100 A degrees C for 1 h. These results suggested that this STAMP has the potential to eliminate MRSA strains without disrupting the normal flora.
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